Ferroptosis inhibitors, antioxidants, compositions for the treatment of intestinal or kidney diseases, foods, and cosmetics, and methods for producing the same.

Abstract

To provide potent antioxidants derived from β-lactoglobulin (β-lactoglobulin derivatives with potent antioxidant activity), ferroptosis inhibitors, antioxidants, therapeutic compositions for intestinal or kidney diseases, foods, and cosmetics utilizing these, as well as methods for producing these. [Solution] A ferroptosis inhibitor, etc., characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

Description

Technical Field

[0001] The present invention relates to a ferroptosis inhibitor, an antioxidant, a therapeutic composition for intestinal diseases or kidney diseases, foods, cosmetics, and methods for producing them.

Background Art

[0002] Beta-lactoglobulin (β-LG), which is a major component of whey protein, is a protein source with excellent safety that can be obtained in large quantities from whey, a by-product derived from the dairy industry. However, many parts of its functions and application possibilities remain not fully understood yet. Also, although β-lactoglobulin is known to have antioxidant ability, its ability is generally considered limited.

[0003] Oxidative stress is a pathological condition in which cell and tissue damage is caused by the overproduction of reactive oxygen species and free radicals. Oxidative stress is deeply involved in the causes and progression of many diseases such as cancer, diabetes, kidney disease, cardiovascular disease, and digestive diseases. However, since there are antioxidant molecules with active reducing power such as glutathione and thioredoxin in the body, usually, oxidative stress does not have a great impact on the living body. However, in such diseases, dysfunction of these active antioxidant defense systems occurs, and as a result, it is considered that oxidative stress causes the onset and progression of diseases.

[0004] The inventors recently found that by reductively cleaving disulfide bonds in structural proteins such as immunoglobulins and albumins, they can be converted into powerful antioxidants (Patent Document 1, Patent Document 2). However, no powerful antioxidant derived from β-lactoglobulin is known.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] The present invention aims to solve the above-mentioned problems and achieve the following objectives. Specifically, the present invention aims to provide a potent antioxidant derived from β-lactoglobulin (a β-lactoglobulin derivative having potent antioxidant activity). [Means for solving the problem]

[0007] As a result of diligent research conducted by the present inventors to achieve the above objective, they have found that it is possible to provide a potent antioxidant derived from β-lactoglobulin (a β-lactoglobulin derivative with potent antioxidant activity), a ferroptosis inhibitor, an antioxidant, a therapeutic composition for intestinal or kidney disease, a food product, and a cosmetic product utilizing this antioxidant, as well as methods for producing these products.

[0008] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are, for example, as follows: <1> This ferroptosis inhibitor is characterized by containing a β-lactoglobulin derivative having two or more thiol groups. <2> The β-lactoglobulin derivative having two or more thiol groups does not have a disulfide bond, <1> It is a ferroptosis inhibitor as described above. <3> This antioxidant is characterized by containing a β-lactoglobulin derivative having two or more thiol groups. <4> The β-lactoglobulin derivative having two or more thiol groups does not have a disulfide bond, <3> It is an antioxidant as described in [reference]. <5> This is a therapeutic composition for intestinal or kidney disease, characterized by containing a β-lactoglobulin derivative having two or more thiol groups. <6> This food product is characterized by containing a β-lactoglobulin derivative having two or more thiol groups. <7> This cosmetic product is characterized by containing a β-lactoglobulin derivative having two or more thiol groups. <8> This is a method for producing a ferroptosis inhibitor, characterized by including treatment of β-lactoglobulin with a reducing agent. <9> This is a method for producing an antioxidant, characterized by including treatment of β-lactoglobulin with a reducing agent. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a potent antioxidant derived from β-lactoglobulin (a β-lactoglobulin derivative having potent antioxidant activity), a ferroptosis inhibitor, an antioxidant, a therapeutic composition for intestinal or kidney disease, a food product, and a cosmetic product utilizing the same, as well as methods for producing these products. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the results of Western blotting using a β-lactoglobulin-specific antibody for whey protein and β-lactoglobulin control in Test Example 1. [Figure 1B] Figure 1B shows the results of quantifying thiol groups using the fluorescent maleimide labeling method (top figure) and the results of EZ blue staining (bottom figure) in Test Example 1. [Figure 1C] Figure 1C is a graph showing the results of quantifying thiol activity using the Elman method in Test Example 1. [Figure 2A] Figure 2A shows the results of quantifying thiol groups using the fluorescent maleimide labeling method (top figure) and the results of EZ blue staining (bottom figure) in Test Example 2. [Figure 2B] Figure 2B is a graph showing the results of quantifying the hydrogen peroxide concentration in Test Example 2. [Figure 2C]Figure 2C shows the thiol / disulfide exchange between reduced β-lactoglobulin and GSH / GSSG in Test Example 2. [Figure 3A] Figure 3A shows the experimental designs for in vivo administration of reduced β-lactoglobulin in Test Examples 3 to 5. [Figure 3B] Figure 3B is a photograph showing the significant improvement in TNBS-induced colonic shortening and rectal bleeding in Test Example 3, achieved by administering reduced β-lactoglobulin. [Figure 3C] Figure 3C is a graph showing the significant improvement in TNBS-induced colonic shortening and rectal bleeding in Study Example 3 with the administration of reduced β-lactoglobulin (mean ± SEM, n=3;** p<0.01 vs. NC;## p<0.01). [Figure 3D] Figure 3D is a graph showing the significant inhibitory effect of administration of reduced β-lactoglobulin on TNBS-induced IL-1α production in the colon in Test Example 3 ((mean ± SEM, n=3;** p<0.01 vs. NC;## p<0.01). [Figure 3E] Figure 3E is a graph showing the significant inhibitory effect of administration of reduced β-lactoglobulin on TNBS-induced TNF-α production in the colon in Test Example 3 (mean ± SEM, n=3;** p<0.01 vs. NC;## p<0.01). [Figure 3F] Figure 3F shows the effect of administering reduced β-lactoglobulin in Test Example 3, which significantly eliminated TNBS-induced lipocalin-2 expression in the colon. [Figure 3G] Figure 3G is a graph showing the quantitative results of the band intensity in Figure 3F (mean ± SEM, n=3; ** p<0.01 vs. NC; ## p<0.01). [Figure 4A]Figure 4A is a graph showing a significant inhibitory effect on the increase in blood urea nitrogen (BUN) induced by TNBS by administration of reduced β-lactoglobulin in Test Example 3 (mean ± SEM, n = 3; **p < 0.01 vs. NC; ##p < 0.01). [Figure 4B] Figure 4B is a graph showing a significant inhibitory effect on the production of IL-1α induced by TNBS in the kidney by administration of reduced β-lactoglobulin in Test Example 3 (mean ± SEM, n = 3; **p < 0.01 vs. NC; ##p < 0.01). [Figure 4C] Figure 4C is a graph showing a significant inhibitory effect on the production of TNF-α induced by TNBS in the kidney by administration of reduced β-lactoglobulin in Test Example 3 (mean ± SEM, n = 3; **p < 0.01 vs. NC; ##p < 0.01). [Figure 4D] Figure 4D is a figure showing the effect of significantly eliminating the expression of lipocalin-2 induced by TNBS in the kidney by administration of reduced β-lactoglobulin in Test Example 3. [Figure 4E] Figure 4E is a graph showing the quantitative results of the band intensity in Figure 4D (mean ± SEM, n = 3; **p < 0.01 vs. NC; ##p < 0.01). [Figure 5A] Figure 5A is a figure showing the results of quantifying thiol groups in the protein lysate of the large intestine by the fluorescent maleimide labeling method in Test Example 4. [Figure 5B] Figure 5B is a graph showing the quantitative results of the band intensity in Figure 5A (mean ± SEM, n = 3; * p < 0.05; ** p < 0.01 vs. NC; # p < 0.05, ## p < 0.01). [Figure 5C] Figure 5C is a figure showing the results of quantifying thiol groups in the protein lysate of the kidney by the fluorescent maleimide labeling method in Test Example 4. [Figure 5D] Figure 5D is a graph showing the quantitative results of the band intensity in Figure 5C (mean ± SEM, n = 3; * p < 0.05; ** p < 0.01 vs. NC; # p < 0.05, ## p < 0.01). [Figure 5E] Figure 5E shows the results of quantifying thiol groups in serum using the fluorescent maleimide labeling method in Test Example 4. [Figure 5F] Figure 5F is a graph showing the quantitative results of the band intensity in Figure 5E (mean ± SEM, n=3; * p<0.05; ** p<0.01 vs. NC; # p<0.05, ## p<0.01). [Figure 6A] Figure 6A shows the Western blot results for SLC7A11, ACSL4, and GPX4 protein lysates from the large intestine in Test Example 5. [Figure 6B] Figure 6B (SLC7A11) is a graph showing the quantitative results of the band intensity in Figure 6A (mean ± SEM, n=3; * p<0.05; ** p<0.01 vs. NC; # p<0.05, ## p<0.01). [Figure 6C] Figure 6C (ACSL4) is a graph showing the quantitative results of the band intensity in Figure 6A (mean ± SEM, n=3; * p<0.05; ** p<0.01 vs. NC; # p<0.05, ## p<0.01). [Figure 6D] Figure 6D (GPX4) is a graph showing the quantitative results of the band intensity in Figure 6A (mean ± SEM, n=3; * p<0.05; ** p<0.01 vs. NC; # p<0.05, ## p<0.01). [Figure 7A] Figure 7A is a photograph showing the results of Calcein-AM / PI staining in Test Example 6. [Figure 7B] Figure 7B is a graph showing the results of the LDH release assay after H2O2 exposure in Test Example 6 (mean ± SEM). [Figure 7C] Figure 7C is a graph showing the results of the LDH release assay after TNBS exposure in Test Example 6 (mean ± SEM). [Figure 7D] Figure 7D shows the results of quantifying thiol groups in cell lysates after H2O2 exposure in Test Example 6 using the fluorescent maleimide labeling method. [Figure 7E]Figure 7E is a graph showing the quantitative results of the blot in Figure 7D. [Figure 7F] Figure 7F shows the results of quantifying thiol groups in cell lysates after TNBS exposure in Test Example 6, using the fluorescent maleimide labeling method. [Figure 7G] Figure 7G shows the quantitative results of the blot from Figure 7F. [Figure 8A] Figure 8A is a photograph showing the results of Calcein-AM / PI staining after TNBS exposure in Test Example 7. [Figure 8B] Figure 8B is a graph showing the results of the LDH release assay after TNBS exposure in Test Example 7. [Figure 8C] Figure 8C is a photograph showing the results of Calcein-AM / PI staining after RSL3 exposure in Test Example 7. [Figure 8D] Figure 8D is a graph showing the results of the LDH release assay after RSL3 exposure in Test Example 7. [Figure 9A] Figure 9A is a photograph showing the changes in intracellular iron ions in Test Example 7, either TNBS-induced (top figure) or RSL3-induced (bottom figure). [Figure 9B] Figure 9B is a graph showing the quantitative results of the image in the upper part of Figure 9A (mean ± SD, n=20; ** P<0.01 vs. control; ## P<0.01). [Figure 9C] Figure 9C is a graph showing the quantitative results of the image in the lower part of Figure 9A (mean ± SD, n=20; ** P<0.01 vs. control; ## P<0.01). [Figure 9D] Figure 9D is a photograph showing the formation of TNBS-induced lipid peroxides in Test Example 7. [Figure 9E] Figure 9E is a graph showing the quantitative results of the images in Figure 9D (mean ± SD, n=20; ** P<0.01 vs. control; ## P<0.01). [Figure 9F] Figure 9F is a photograph showing the RSL-3-induced formation of lipid peroxides in Test Example 7. [Figure 9G] Figure 9G is a graph showing the quantitative results of the images in Figure 9F (mean ± SD, n=20; ** P<0.01 vs. control; ## P<0.01). [Figure 10A] Figure 10A shows the results of a Western blot regarding SOH levels and P38 activation in Test Example 8. [Figure 10B] Figure 10B shows the quantitative results of the blot (SOH level) from Figure 10A (mean ± SE, n=3, ** P<0.01 vs. control; # P<0.01). [Figure 10C] Figure 10C shows the quantitative results of the blot (P38 activation) from Figure 10A (mean ± SE, n=3, ** P<0.01 vs. control; # P<0.01). [Figure 10D] Figure 10D shows the ELISA results for IL-1β in Test Example 8 (mean ± SE, n=3, ** P<0.01 vs. control; # P<0.01). [Figure 10E] Figure 10E shows the ELISA results for TNFα in Test Example 8 (mean ± SE, n=3, ** P<0.01 vs. control; # P<0.01). [Modes for carrying out the invention]

[0011] (Ferroptosis inhibitors) The ferroptosis inhibitor comprises a β-lactoglobulin derivative having two or more thiol groups, and may further contain other components.

[0012] -β-lactoglobulin derivative having 2 or more thiol groups- Beta-lactoglobulin (sometimes referred to as "conventional beta-lactoglobulin") is a major component of whey protein. The aforementioned β-lactoglobulin has two disulfide bonds (sometimes referred to as "SS bonds") and one thiol group (sometimes referred to as "SH group," "sulfanyl group," "hydrulfur group," or "sulfhydryl group") within its molecule.

[0013] There are no particular restrictions on the source of the β-lactoglobulin, and it can be selected as appropriate; for example, it can come from mammals such as cattle. Bovine β-lactoglobulin is a 162-residue protein with a molecular weight of 18.4 kDa (kilodaltons).

[0014] There are no particular restrictions on the β-lactoglobulin derivative having two or more thiol groups, and they can be selected as appropriate. However, β-lactoglobulin derivatives having three or more thiol groups are preferred, β-lactoglobulin derivatives having four or more thiol groups are more preferred, and β-lactoglobulin derivatives having five thiol groups are even more preferred. In other words, it is preferable that the β-lactoglobulin derivative having two or more thiol groups does not have a disulfide bond.

[0015] The β-lactoglobulin derivative having two or more thiol groups is sometimes referred to as "reduced β-lactoglobulin."

[0016] The reduced β-lactoglobulin has excellent ferroptosis suppression and excellent antioxidant activity.

[0017] Here, among the β-lactoglobulin derivatives having three or more thiol groups, the β-lactoglobulin derivative having three thiol groups has one disulfide bond in the β-lactoglobulin molecule replaced by a free thiol group. Furthermore, in the β-lactoglobulin derivative having the five thiol groups, two disulfide bonds within the β-lactoglobulin molecule are replaced with free thiol groups. In the β-lactoglobulin derivative having two or more thiol groups, some of the thiol groups may be SOH groups, but it is preferable that they do not have SOH groups.

[0018] There are no particular restrictions on the content of the β-lactoglobulin derivative having two or more thiol groups in the ferroptosis inhibitor, and it can be appropriately selected depending on the purpose. The ferroptosis inhibitor may consist only of the β-lactoglobulin derivative having two or more thiol groups.

[0019] -Other ingredients- The aforementioned other components are not particularly limited as long as they do not impair the effects relating to this disclosure, and components used in known pharmaceuticals, foods, cosmetics, etc. can be appropriately selected according to the dosage form, etc. Examples include nonionic surfactants, sugars, sugar alcohols, polysaccharides, polyacrylic acid, polyethylene oxide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethylcellulose, hydroxypropylcellulose, and starch. These may be used individually or in combination of two or more.

[0020] There are no particular restrictions on the content of the other components in the ferroptosis inhibitor, and they can be appropriately selected depending on the purpose.

[0021] The ferroptosis inhibitor may be used alone or in combination with a pharmaceutical product containing other active ingredients. Furthermore, the ferroptosis inhibitor may be used in combination with a pharmaceutical product containing other active ingredients.

[0022] There are no particular restrictions on the manner in which the ferroptosis inhibitor is used, and it can be appropriately selected according to the purpose. Examples include oral, transdermal, enteral, transmucosal, transvenous, transarterial, subcutaneous, and intramuscular administration. These may be used individually or in combination of two or more.

[0023] There are no particular restrictions on the dosage form of the ferroptosis inhibitor, and known dosage forms can be appropriately selected depending on the manner of use. The dosage form may be solid, semi-solid, or liquid, and examples include oral preparations such as tablets, granules, powders, capsules, soft capsules, and syrups; transdermal or transmucosal preparations such as liquids, ointments, creams, gels, sprays, patches, inhalants, and suppositories; and injectable preparations. The ferroptosis inhibitor of the aforementioned dosage form can be manufactured by known methods.

[0024] There are no particular restrictions on the dosage of the ferroptosis inhibitor administered to an individual; it can be appropriately selected considering the age, weight, and presence or absence of disease of the individual being administered. For example, a single dose may be 0.05 to 20 g / kg (grams per kilogram).

[0025] There are no particular restrictions on the administration interval of the ferroptosis inhibitor; it can be appropriately selected considering the age, weight, presence or absence of disease, etc., of the individual being treated.

[0026] There are no particular restrictions on the individuals targeted by the ferroptosis inhibitor, and they can be appropriately selected depending on the purpose. Examples include humans, mice, rats, cattle, pigs, monkeys, dogs, and cats.

[0027] There are no particular restrictions on the diseases that the ferroptosis inhibitors can target; they can be appropriately selected depending on the purpose. Examples include diseases caused by oxidative stress, intestinal diseases, and kidney diseases. Diseases caused by oxidative stress include, for example, Alzheimer's disease, Parkinson's disease, complications of diabetes, and rheumatoid arthritis. Among these, Alzheimer's disease, Parkinson's disease, complications of diabetes, rheumatoid arthritis, intestinal diseases, and kidney diseases are particularly suitable.

[0028] (Antioxidant) The antioxidant comprises a β-lactoglobulin derivative having two or more thiol groups, and may further contain other components. The β-lactoglobulin derivative having two or more thiol groups, and the other components are as described above in "(ferroptosis inhibitors)".

[0029] The antioxidant may be used alone or in combination with a pharmaceutical product containing other active ingredients. Furthermore, the antioxidant may be used in a state where it is incorporated into a pharmaceutical product containing other active ingredients.

[0030] The mode of use, dosage form, dosage to be administered to an individual, administration interval, target individuals, and target diseases of the aforementioned antioxidant are as described above in "(ferroptosis inhibitors)".

[0031] (Composition for the treatment of intestinal or kidney diseases) The aforementioned therapeutic composition for intestinal or kidney disease comprises a β-lactoglobulin derivative having two or more thiol groups, and may further contain other components. The β-lactoglobulin derivative having two or more thiol groups, and the other components are as described above in "(ferroptosis inhibitors)".

[0032] The aforementioned therapeutic composition for intestinal or kidney disease may contain a ferroptosis inhibitor or an antioxidant. The ferroptosis inhibitors are as described above in "(ferroptosis inhibitors)". The aforementioned antioxidants are as described above under "(Antioxidants)". There are no particular restrictions on the amount of the ferroptosis inhibitor or antioxidant in the therapeutic composition for intestinal or kidney disease, and it can be appropriately selected depending on the purpose.

[0033] The aforementioned therapeutic composition for intestinal or kidney disease may be used alone or in combination with a pharmaceutical product containing other active ingredients. Furthermore, the therapeutic composition for intestinal or kidney disease may be used in combination with a pharmaceutical product containing other active ingredients.

[0034] The mode of use, dosage form, dosage to administer to an individual, administration interval, target individual, and target disease of the aforementioned therapeutic composition for intestinal or kidney disease are as described above in "(ferroptosis inhibitor)".

[0035] (food) The aforementioned food contains a β-lactoglobulin derivative having two or more thiol groups, and may further contain other components. The β-lactoglobulin derivative having two or more thiol groups, and the other components are as described above in "(ferroptosis inhibitors)".

[0036] The aforementioned food may contain a ferroptosis inhibitor or an antioxidant. The ferroptosis inhibitors are as described above in "(ferroptosis inhibitors)". The aforementioned antioxidants are as described above under "(Antioxidants)". There are no particular restrictions on the amount of the ferroptosis inhibitor or antioxidant in the food product, and they can be appropriately selected depending on the purpose.

[0037] There are no particular restrictions on the type of food, and any known food can be appropriately selected. In the present invention, the food can be anything that can be taken orally, and includes, for example, general food and beverages, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for sick people. Beverages are also included. As described above, the food of the present invention contains reduced β-lactoglobulin, which has excellent ferroptosis suppression and excellent antioxidant activity, and can therefore be suitably used as an anti-aging food and the like.

[0038] (cosmetics) The cosmetic product contains a β-lactoglobulin derivative having two or more thiol groups, and may further contain other ingredients. The β-lactoglobulin derivative having two or more thiol groups, and the other components are as described above in "(ferroptosis inhibitors)".

[0039] The aforementioned cosmetic may contain a ferroptosis inhibitor or an antioxidant. The ferroptosis inhibitors are as described above in "(ferroptosis inhibitors)". The aforementioned antioxidants are as described above under "(Antioxidants)". There are no particular restrictions on the amount of the ferroptosis inhibitor or antioxidant contained in the cosmetic product, and they can be appropriately selected depending on the purpose.

[0040] There are no particular restrictions on the type of cosmetic product, and any known cosmetic product can be appropriately selected, such as emulsions, creams, lotions, masks, serums, cleansers, and makeup products. As described above, the cosmetic product of the present invention contains reduced β-lactoglobulin, which has excellent ferroptosis suppression and excellent antioxidant properties, and is therefore suitable for use as an anti-aging cosmetic product.

[0041] When the reduced β-lactoglobulin is administered, it is oxidized in the oxidative environment of the body and forms the same structure as normal β-lactoglobulin. Therefore, according to the ferroptosis inhibitor, antioxidant, therapeutic composition for intestinal or kidney disease, food, or cosmetic according to this disclosure, it is possible to exert not only the antioxidant effect of reduced β-lactoglobulin but also the function of normal β-lactoglobulin, making it applicable to a wide range of diseases.

[0042] (Method for manufacturing ferroptosis inhibitors) The method for producing the ferroptosis inhibitor includes a reduction step and may further include other steps.

[0043] -Reduction Process- The reduction step described above is a step of treating β-lactoglobulin (normal β-lactoglobulin) with a reducing agent. The reduction step is a step to obtain reduced β-lactoglobulin.

[0044] The β-lactoglobulin (conventional β-lactoglobulin) may be commercially available β-lactoglobulin or one that has been prepared as appropriate.

[0045] The reducing agent is not particularly limited as long as it can cleave the disulfide bond in the conventional β-lactoglobulin and form a thiol group in the β-lactoglobulin, and can be appropriately selected depending on the purpose. Examples include dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), and hydrogen sulfide (H2S). These may be used individually or in combination of two or more.

[0046] The reducing agent may be a commercially available reducing agent or one that has been prepared as appropriate.

[0047] There are no particular restrictions on the reduction (reduction treatment) method, and it can be appropriately selected according to the purpose. For example, one method is to add the reducing agent to a solution containing the conventional type β-lactoglobulin and allow it to react. There are no particular restrictions on the amounts of each component, temperature, time, and other conditions in the above reaction; they can be appropriately selected considering the degree of reduction, etc. The solution after the reduction (reduction treatment) may be purified by filtration or dialysis, if necessary.

[0048] -Other processes- The aforementioned other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, one example is a step of mixing the reduced β-lactoglobulin with the other components mentioned above.

[0049] (Method of manufacturing antioxidants) The method for producing the antioxidant includes a reduction step and may further include other steps. The reduction step and the other steps are as described in the above-mentioned "(Method for producing a ferroptosis inhibitor)".

[0050] (Method for producing a therapeutic composition for intestinal or kidney disease) The method for producing the therapeutic composition for intestinal or kidney disease includes a reduction step and may further include other steps. The reduction step and the other steps are as described in the above-mentioned "(Method for producing a ferroptosis inhibitor)".

[0051] When the composition for treating intestinal or kidney disease contains a ferroptosis inhibitor or an antioxidant, there are no particular restrictions on the method for producing the composition for treating intestinal or kidney disease, as long as it can contain the ferroptosis inhibitor or the antioxidant, and a known method can be appropriately selected depending on the type of cosmetic.

[0052] (Food manufacturing methods) The method for producing the food product may include a reduction step and may further include other steps. The reduction step and the other steps are as described in the above-mentioned "(Method for producing a ferroptosis inhibitor)".

[0053] When the food contains a ferroptosis inhibitor or an antioxidant, there are no particular restrictions on the method for producing the food, as long as the ferroptosis inhibitor or antioxidant can be included, and a known method can be appropriately selected depending on the type of cosmetic.

[0054] (Method of manufacturing cosmetics) The method for manufacturing the cosmetic product may include a reduction step and may further include other steps. The reduction step and the other steps are as described in the above-mentioned "(Method for producing a ferroptosis inhibitor)".

[0055] When the cosmetic product contains a ferroptosis inhibitor or an antioxidant, there are no particular restrictions on the method for manufacturing the cosmetic product, as long as the ferroptosis inhibitor or antioxidant can be included, and a known method can be appropriately selected depending on the type of cosmetic product. [Examples]

[0056] The embodiments relating to the present disclosure described above will be explained below, but the present invention described using the present disclosure as an example is not limited in any way to these embodiments.

[0057] 1. Experimental materials and methods (1 material) The β-lactoglobulin protein powder was purchased from Sigma Aldrich (St. Louis, MO, USA). Dimedone and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). I purchased Alexa 680 Fluor C2 Maleimide from Thermo Scientific (Rockford, IL, USA). HRP-labeled anti-rabbit or mouse IgG, anti-β-actin, anti-GPX4, and anti-caspase-3 antibodies were purchased from Cell Signaling (Beverly, MA, USA). The anti-SLC7A11(xCT) antibody was purchased from Novus Biologicals USA (CO, USA). The β-lactoglobulin antibody was purchased from GeneTex (North America). The blood urea nitrogen (BUN) kit was purchased from Thermo Fisher Scientific (Frederick, MD, USA). The anti-lipocalin-2 antibody was obtained from Adipogen (MI, USA). E-cadherin was purchased from BD Biosciences (NJ, USA). The interleukin-1α, interleukin-1β, and tumor necrosis factor-α assay kits were purchased from PeproTech (Rocky Hill, NJ, USA). ROS and superoxide were purchased from Enzo Life Sciences (NY, USA), hydrogen peroxide (H2O2) detection kit, (1S,3R)-RSL-3, reproxistatin-1, and deferoxamine (DFO) from Cayman Chemical Company (MI, USA), and calcein-AM and propidium iodide (PI) staining kit, FerroOrange reagent, and lipid peroxide detection kit from Dojin Chemical Laboratories Co., Ltd. (Japan). Pentobarbital sodium was purchased from Kyoritsu Pharmaceutical Co., Ltd. (Tokyo, Japan). Maleimide, GSH, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0058] (2. Animal experiments) Mature male and female C57BL / 6 mice weighing 20 to 30 g were housed in a 12-hour light-dark cycle with free access to standard solid food and water. All animal procedures were approved by the Yamanashi University Animal Experimentation Committee and carried out in accordance with institutional and national guidelines regarding the ethical treatment of animals.

[0059] To induce TNBS colitis, mice were rectally administered 200 mg / kg of TNBS (Sigma-Aldrich) dissolved in 50% ethanol. Control mice were administered physiological saline. Prior to TNBS administration, mice were anesthetized by intraperitoneal injection of 50 to 100 mg / kg of pentobarbital.

[0060] After administering TNBS and allowing the mice to stand for 180 seconds, they were returned to their cages and observed daily for weight loss, diarrhea, and rectal bleeding for 1–3 days. At the experimental endpoint, mice were euthanized with pentobarbital, and blood, kidney, and colon samples were collected for macroscopic scoring of inflammation, oxidation, and damage, histological analysis, and other evaluations.

[0061] In animal experiments, mice were divided into five experimental groups (n=4 / group): a negative control group (untreated: NC), a TNBS-positive control group (Ctrl), a TNBS-treated control group (administered with conventional β-lactoglobulin: β-LG), a TNBS-treated group (administered with reduced β-lactoglobulin: r-β-LG), and a TNBS-treated group (inhibited) (administered with maleimide-treated reduced β-lactoglobulin: blocked r-β-LG). Experiments using the same protocol were repeated at least twice, and representative results were shown.

[0062] (3 Caco-2 cell culture) Caco-2 cells (ATCC) were maintained in DMEM / F12 medium supplemented with 5% fetal bovine serum and 1% antibiotic / antifungal solution in a 37°C, 5% CO2 incubator. In the experiment, cells were seeded on cell culture plates in medium containing 0.5% fetal bovine serum (FBS) and processed as specified.

[0063] (4. Primary culture of bone marrow-derived macrophages (BMDMs)) BMDMs were obtained from mouse femurs. Specifically, bone marrow cells were obtained from mouse femurs using DMEM / F12 containing antibiotics. More precisely, the epiphysis of the femur was removed, and bone marrow cells were obtained by flushing the bone cavity with a syringe fitted with a 21G needle.

[0064] Collected bone marrow cells were seeded in 12-well plates at a density of 1.5 to 2 million cells per well and cultured in DMEM / F12 supplemented with 20% FBS and 40 ng / mL macrophage colony-stimulating factor (M-CSF). After 2 days, the culture medium was changed. After a further 3 days of culture, non-adherent cells were removed, and adhered BMDMs were used in the experiment.

[0065] (5. Western blot analysis) Western blot analysis was performed as follows: Cell and tissue lysates were prepared using SDS and RIPA cleavage buffer, respectively, and supplemented with a protease inhibitor cocktail. Protein concentration was measured using a Micro BCA assay (Thermo Fisher Scientific).

[0066] Equal amounts of protein were separated on a 10% gel by SDS-PAGE, transferred to a PVDF membrane, and blocked with Tris-buffered saline (TBST) containing 0.1% Tween-20 with 5% skim milk or 3% BSA. At 4°C overnight, primary antibodies (β-lactoglobulin (GTX77272, GeneTex); caspase-3 (cleaved caspase-3) (9662, Cell Signaling); β-actin (A5316, Sigma); lipocalin-2 (AG-25A-0088, Adipogen); p-P38 (9662, Cell Signaling); E-cadherin (sc-7870, Santa Cruz Biotechnology); NOX-2 (bs-3889R, Bioss); NOX-4 (GTX121929, GeneTex); GPX4 (52455, Cell Signaling); SLC7A11 (NB300-317, Novus) The sample was reacted with Biologicals or ACLS4 (22401-1-AP, Proteintech), and then with HRP-labeled secondary antibody (7776S or 7074S, Cell Signaling) for 1 hour.

[0067] Immunoreaction bands were visualized using sensitized chemiluminescence (Nacalai Tesque) and imaged using the Fujifilm LAS-1000 system. Band density was quantified using NIH ImageJ software. Equivalent protein loading was verified by β-actin immunoblotting or EZ blue staining.

[0068] (6. Detection of free thiol groups (free sulfhydryl groups) (-SH) by fluorescent maleimide labeling method) Various whey protein (WP) samples were reacted with 5 μM Alexa Fluor 680 C2 maleimide (far-red fluorescence) at 4°C. Labeled proteins were separated by SDS-PAGE, and in-gel fluorescence was imaged using a Fujifilm LAS-1000 system. The intensity of the fluorescence bands was quantified using ImageJ software and normalized by the EZ blue-stained protein bands indicating the fluorescent protein loading in each lane.

[0069] (7. Lactate dehydrogenase (LDH) assay) Cell damage was quantified using the Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (Takara Bio) according to the manufacturer's protocol. Cells were seeded in 96-well plates and grown in Dulbecco's Modified Eagle Medium / Mix F-12 (DMEM / F12) supplemented with fetal bovine serum (FBS) until they reached 80 to 90% confluence. After stimulation, the cell culture supernatant was collected and reacted with an equal volume of the assay buffer provided in the kit for 30 minutes at room temperature. LDH release was quantified by measuring absorbance at 490 nm (nanometers). Cytotoxicity was calculated as the ratio of released LDH to total LDH release. Complete cell lysates using 2% Triton X-100 were used to determine total LDH release. Values ​​from cell culture medium alone were used as a background control.

[0070] (8 Enzyme-linked immunosorbent assay (ELISA)) Cytokine measurements by ELISA were performed as follows. Colon and kidney tissue (40 mg) was homogenized with 200 μL of RIPA buffer to obtain tissue lysates. The tissue lysates were centrifuged, and IL-1α, IL-1β, and TNF-α were quantified from the supernatant according to the protocol of the manufacturer of the ELISA kit (Peprotech, Rocky Hill, NJ, USA).

[0071] (9. Calcein-AM and propidium iodide (PI) staining) Cell viability was evaluated using the Calcein-AM / PI viability staining kit (Dojin Chemical Laboratories Co., Ltd.) according to the kit's instructions. The cells were reacted with a mixture of Calcein-AM and PI at 37°C for 10 to 20 minutes. Calcein-AM penetrates living cells and is converted to green fluorescent calcein by intracellular esterases. On the other hand, PI penetrates only damaged cell membranes and stains nuclear DNA red. Using a fluorescence microscope, living cells (green) and dead cells (red) were visualized and photographed.

[0072] (10. Measurement of blood urea nitrogen (BUN)) Serum BUN levels were determined using a commercially available colorimetric quantification kit, following the instructions of the kit manufacturer. Serum samples were diluted as needed and reacted with the assay reagent at room temperature for 30 minutes. The optical density at 450 nm was measured using a spectrophotometer (SpectraMax 340).

[0073] (11. Modification (derivativeization) of β-lactoglobulin protein using the reducing chemical DTT) The low-temperature processed β-lactoglobulin powder was purchased from Sigma-Aldrich (Cat# L0130-5G, CO, USA). The aforementioned powder (4.5 g / 30 mL DW) was reconstituted in distilled water to 150 mg / mL (milligrams per milliliter) and used for reductive modification and subsequent experiments. The β-lactoglobulin solution was reacted overnight at 4°C in the presence of 100 mM (millimolar) dithiothreitol (DTT), and then thoroughly dialyzed with distilled water or 0.9% saline using a 3500 MWCO dialysis tube (Thermo Scientific). Some of the proteins were further modified overnight at 4°C in the presence of 50 mM maleimide before removing unresponsive chemicals by dialysis. The protein concentration of the prepared solution was measured, the free sulfhydryl content was analyzed, and the solutions were aliquoted and stored at -80°C until use.

[0074] (12. Analysis of the hydrogen peroxide (H2O2) scavenging ability of modified β-lactoglobulin (reduced β-lactoglobulin)) The hydrogen peroxide (H2O2) scavenging ability of modified β-lactoglobulins was evaluated by measuring the change in H2O2 concentration before and after reaction using Cayman Chemical Company's H2O2 assay kit (Cat# 600050; Ann Arbor, MI, USA) for different modified β-lactoglobulin samples.

[0075] Specifically, a 1 mM H2O2 solution was reacted for 1 hour in the presence or absence of each modified β-lactoglobulin sample at a concentration of 2 mg / mL. After the reaction, 40 μL of each sample was transferred to a microplate well, mixed, and reacted in the dark with the assay buffer and enzyme reaction solution provided in the kit at room temperature for 30 minutes. To quantify the remaining H2O2 concentration, fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

[0076] (13 Detection of ROS production) Superoxide anion and reactive oxygen species (ROS) levels were measured using the kit (Cat#ENZ-51010, Enzo Life Sciences, Farmingdale, NY, USA) according to the kit manufacturer's protocol. Cells were seeded in 96-well plates and preloaded for 3 hours with superoxide detection reagent (orange fluorescence) and oxidative stress detection reagent (green fluorescence). Next, they were stimulated with LPS or TNBS for 1 hour, either alone or in combination. Fluorescence images were acquired using a fluorescence microscope (IX71, Olympus Corporation, Tokyo, Japan) to visualize superoxide and ROS levels through orange and green fluorescence, respectively.

[0077] (14 Intracellular divalent iron ions (Fe 2+ )Verification) Intracellular Fe2+ The levels were evaluated using FerroOrange fluorescent probes (product number F374) obtained from Dojin Chemical Laboratories Co., Ltd. After processing, the cell samples were washed and incubated in 1 μM FerroOrange solution at 37°C for 30 minutes. A fluorescence microscope was used to acquire images, and the obtained images were processed and analyzed using ImageJ software. The average fluorescence intensity of each experimental cohort was standardized relative to the control group.

[0078] (15. Evaluation of lipid peroxidation) Lipid peroxidation was evaluated using the fluorescent probe BDP 581 / 591 C11 (catalog number L267, Dojin Chemical Research Institute Co., Ltd.). After the treatment experiment, cell specimens were reacted in fresh culture medium with 2.5 μM BDP 581 / 591 C11 staining solution at 37°C for 30 minutes. After this reaction period, the cells were washed before being photographed under a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software, and the average fluorescence intensity observed in each experimental group was appropriately standardized relative to the control group.

[0079] (16 statistical analysis) The data is presented as mean ± standard error (SEM). Statistical comparisons between the two groups were performed using unpaired Student's t-tests. For comparisons of multiple groups with a common control, a one-way analysis of variance (ANOVA) was performed using Tukey's post-hoc test. All analyses were performed using Microsoft Excel or GraphPad Prism version 8.0. A value of p < 0.05 was considered statistically significant.

[0080] 2. Example Test (Test Example 1: Reduction treatment of β-lactoglobulin and quantification of thiol groups) First, we confirmed that whey protein (WP) contains β-lactoglobulin protein. As shown in Figure 1A, when Western blotting was performed using a β-lactoglobulin-specific antibody, a band was detected in the lane loaded with whey protein (WP) at the same position as in the lane loaded with β-lactoglobulin control (β-LG), indicating that whey protein contains β-lactoglobulin protein (normal β-lactoglobulin).

[0081] Next, in order to develop β-lactoglobulin as a thiol antioxidant (reduced β-lactoglobulin), the disulfide bond was cleaved to expose the free thiol group. Specifically, β-lactoglobulin was reductively treated with 100 mM DTT.

[0082] Figures 1B and 1C show that the reduction treatment significantly increased the binding of β-lactoglobulin to the maleimide-reactive fluorescent probe.

[0083] The upper part of Figure 1B shows the results of quantifying thiol groups using the fluorescent maleimide labeling method. Compared to normal β-lactoglobulin (β-LG), reduced β-lactoglobulin (r-β-LG) showed an increase in thiol groups, and this increase was inhibited by maleimide (blocked r-β-LG). The lower part of Figure 1B shows the results of EZ blue staining, confirming that an equal amount of protein was loaded into each lane. The observation of an upward shift in the band following the reduction treatment suggested the cleavage of an intramolecular disulfide bond.

[0084] As shown in Figure 1C, consistent with the above results, the reduction treatment increased thiol activity (-SH activity) as shown by the Elman method. This increase was completely inhibited by pretreatment of the thiol group (-SH group) of reductively treated β-LG (r-β-LG) with unlabeled maleimide. These results indicate that the reduction treatment increased the number and activity of thiol groups in β-LG.

[0085] Based on these results, it was confirmed by fluorescent maleimide labeling and the Elman method that β-lactoglobulin (reduced β-lactoglobulin) after reduction treatment possesses high levels of free thiol groups and activity.

[0086] (Test Example 2: Antioxidant Activity) To determine whether the increased thiol groups in reduced β-lactoglobulin possess antioxidant activity, we investigated the interaction of reduced β-lactoglobulin with hydrogen peroxide and GSH / GSSG, which are major oxidizing agents.

[0087] As shown in the upper part of Figure 2A, in the fluorescent maleimide labeling method, the number of free thiol groups in reduced β-lactoglobulin decreased upon reaction with hydrogen peroxide. Furthermore, the upward band shift that occurred after the reduction treatment disappeared after the reaction with hydrogen peroxide, suggesting that the original protein structure was restored through the reformation of disulfide bonds.

[0088] The lower part of Figure 2A shows the results of EZ blue staining, confirming that an equal amount of protein was loaded into each lane.

[0089] Specifically, 5 μg (micrograms) of conventional β-lactoglobulin or reduced β-lactoglobulin was reacted with 10 mM hydrogen peroxide for 1 hour, and then the structure of β-lactoglobulin was analyzed by Western blotting using a portion of the product (1 μg). The disappearance of the upward shift of reduced β-lactoglobulin (reduced β-lactoglobulin) after reaction with hydrogen peroxide suggests that the original structure was reconstructed.

[0090] Next, we investigated whether the interaction between reduced β-lactoglobulin (reduced β-lactoglobulin) and hydrogen peroxide resulted in a decrease in hydrogen peroxide concentration. Specifically, 2 mg / mL of conventional β-lactoglobulin or reduced β-lactoglobulin was reacted with 1 mM hydrogen peroxide for 1 hour, and then the hydrogen peroxide concentration was detected.

[0091] As shown in Figure 2B, reacting hydrogen peroxide with reduced β-lactoglobulin significantly reduced the hydrogen peroxide concentration. This effect was thiol group-dependent and was not observed in reduced β-lactoglobulin in which the thiol groups were blocked with maleimide.

[0092] The glutathione system is the primary thiol antioxidant system in living organisms. The GSH / GSSG ratio is used as a marker reflecting the in vivo redox state. Therefore, we investigated whether a modified protein (reduced β-lactoglobulin) directly interacts with GSH / GSSG.

[0093] Conventional β-lactoglobulin or reduced β-lactoglobulin was reacted with GSH or GSSG, and the changes in the thiol groups of β-lactoglobulin were measured using a fluorescent maleimide probe.

[0094] Specifically, 3 mg / mL of β-lactoglobulin (either conventional β-lactoglobulin or reduced β-lactoglobulin) was reacted overnight with 2 mM GSH or 2 mM GSSG, respectively. A portion of the reaction product (150 μg) was removed by TCA / acetone precipitation to remove small molecules and unreacted reagents, then prepared to 1 μg / μL in SDS sample buffer. 20 μL of this solution was reacted with fluorescent maleimide for 30 minutes, and the fluorescence signal was detected after separation on the SDS gel.

[0095] As shown in Figure 2C, when reduced β-lactoglobulin was reacted with GSH or GSSG, a change occurred in the thiol group level of the reduced β-lactoglobulin. GSH increased the number of thiol groups in conventional β-lactoglobulin, suggesting exposure of disulfide bonds. Conversely, GSSG decreased the number of thiol groups in reduced β-lactoglobulin, suggesting oxidation induction of thiol groups, and possibly reformation of disulfide bonds. All of these changes were not observed by pretreatment of the thiol groups with unlabeled maleimide. These results indicate that thiol / disulfide exchange exists between reduced β-lactoglobulin and GSH / GSSG.

[0096] These results suggest that reduced β-lactoglobulin interacts with hydrogen peroxide and GSSG via its thiol groups (i.e., reduced β-lactoglobulin scavenges hydrogen peroxide and interacts with the GSH / GSSG system). This indicates that reduced β-lactoglobulin may confer antioxidant activity and link to the thiol antioxidant system.

[0097] (Test Example 3: Efficacy against TNBS-induced colonic and renal impairment) We investigated whether in vivo administration of reduced β-lactoglobulin could protect mice from oxidative stress-centered diseases. Specifically, the effects of oral administration of conventional β-lactoglobulin or reduced β-lactoglobulin on TNBS-induced colonic and renal disorders were evaluated (Figures 3 and 4).

[0098] Figure 3A shows the experimental design for in vivo administration of reduced β-lactoglobulin. Mice were orally administered 1 g / kg of either conventional or reduced β-lactoglobulin five times at 12-hour intervals before and after TNBS-induced colitis.

[0099] In vivo administration experiments were conducted on five experimental groups (n=4 / group): a negative control group (untreated: NC), a TNBS-positive control group (Ctrl), a TNBS-treated control group (administered with conventional β-lactoglobulin: β-LG), a TNBS-treated group (administered with reduced β-lactoglobulin: r-β-LG), and a TNBS-treated group (inhibited) (administered with maleimide-treated reduced β-lactoglobulin: blocked r-β-LG).

[0100] As shown in Figures 3B and 3C, TNBS caused colonic shortening and bleeding, but administration of reduced β-lactoglobulin significantly prevented and dramatically improved TNBS-induced colonic shortening and rectal bleeding. As shown in Figures 3D (IL-1α) and 3E (TNF-α), it was also found that inflammatory cytokine production induced by TNBS in the colon was reduced by administration of reduced β-lactoglobulin. As shown in Figures 3F and 3G, it was also found that the expression of lipocalin-2, a damage marker (colon injury marker) induced by TNBS in the colon, disappears upon administration of reduced β-lactoglobulin. Figure 3G shows the quantitative results of the band intensity in Figure 3F.

[0101] These results indicate that administration of reduced β-lactoglobulin reduces colitis and cytotoxicity.

[0102] Furthermore, as shown in Figure 4A, the increase in blood urea nitrogen (BUN) induced by TNBS was significantly suppressed by the administration of reduced β-lactoglobulin, confirming a protective effect against renal impairment. As shown in Figures 4B (IL-1α) and 4C (TNF-α), it was also found that inflammatory cytokine production induced by TNBS in the kidney was reduced by administration of reduced β-lactoglobulin. As shown in Figures 4D and 4E, it was also found that the expression of lipocalin-2, a damage marker (renal impairment marker) induced by TNBS in the kidney, disappears upon administration of reduced β-lactoglobulin. Figure 4E shows the quantitative results of the band intensity in Figure 4D.

[0103] From these results, it was found that administration of reduced β-lactoglobulin reduces renal impairment.

[0104] (Test Example 4: Effect on improving local and systemic oxidative state) Given that oxidative stress plays a crucial role in the onset and progression of colitis and renal impairment, the protective effect of in vivo administration of reduced β-lactoglobulin may be due to its regulatory effects on local and systemic redox states. Therefore, to evaluate the effects of reduced β-lactoglobulin on local and systemic redox states, levels of thiol groups were measured in the colon, kidneys, and serum.

[0105] Specifically, the thiol group levels of proteins were analyzed for the following samples from the five experimental groups using fluorescent maleimide labeling: intestinal protein lysates (Figures 5A and 5B), kidney protein lysates (Figures 5C and 5D), and serum (Figures 5E and 5F). 10 μg of tissue lysate or 1:100 diluted serum was reacted with 5 mM fluorescent maleimide for 30 minutes.

[0106] As shown in Figures 5A to 5F, administration of reduced β-lactoglobulin effectively reversed the TNBS-induced decrease in protein thiol group levels in the colon, kidney, and serum, indicating that it readjusts the systemic and local oxidative state. Figure 5B shows the quantitative results of the band intensity in Figure 5A, Figure 5D shows the quantitative results of the band intensity in Figure 5C, and Figure 5F shows the quantitative results of the band intensity in Figure 5E.

[0107] Based on these results, it was found that the therapeutic effect of reduced β-lactoglobulin is associated with the improvement of local and systemic oxidative stress.

[0108] (Test Example 5: Effects on ferroptosis) Ferroptosis is mediated by the accumulation of iron-dependent lipid reactive oxygen species and is suggested to be involved in the development of various diseases, particularly intestinal diseases. Molecules such as cystine transporter (SLC7A11), long-chain fatty acid CoA ligase 4 (ACSL4), and glutathione peroxidase 4 (GPX4) play important roles in the development of ferroptosis and are therefore widely used as ferroptosis markers.

[0109] Therefore, we investigated the effects of reduced β-lactoglobulin therapy (in vivo administration) on TNBS-induced changes in these molecules in the colon. Specifically, Western blot analysis of SLC7A11, ACSL4, and GPX4 was performed using colon protein lysates from the five experimental groups mentioned above.

[0110] As shown in Figure 6A, TNBS-induced colitis was associated with decreased levels of SLC7A11 and GPX4, which indicate ferroptosis induction. Unexpectedly, TNBS administration did not have a significant effect on ACSL4. Furthermore, in vivo oral administration of reduced β-lactoglobulin significantly suppressed ferroptosis. In other words, reduced β-lactoglobulin treatment prevented the decline of TNBS-induced SLC7A11 and GPX4.

[0111] Interestingly, reduced β-lactoglobulin not only simply prevented the effects of TNBS on GPX4 and ACSL4, but also appeared to elevate GPX4 beyond basal levels and suppress ACSL4, suggesting that it may possess unique additional effects on these molecules. Figures 6B (SLC7A11), 6C (ACSL4), and 6D (GPX4) show the quantitative results of the band intensities in Figure 6A.

[0112] These results demonstrate the important involvement of ferroptosis in TNBS-induced colitis and its prevention by reduced β-lactoglobulin, showing that reduced β-lactoglobulin attenuates TNBS-induced colonic ferroptosis.

[0113] (Test Example 6: Effects of H2O2 and TNBS-induced oxidative cytotoxicity in colonic epithelial cells) To further investigate the mechanism and role of reduced β-lactoglobulin in TNBS-induced colorectal cell damage, we observed the effects of reduced β-lactoglobulin on H2O2 and TNBS-induced colorectal cell damage in the presence of conventional β-lactoglobulin or reduced β-lactoglobulin, or in the presence of the thiol antioxidant GSH.

[0114] Specifically, Caco-2 cells were exposed to 500 μM H2O2 or 1 mg / mL TNBS for 6 hours (H2O2) or 24 hours (TNBS) in the presence of 5 mg / mL conventional or reduced β-lactoglobulin, or 5 mM thiol antioxidant GSH, and cell death was evaluated by calcein-AM / PI staining (Figure 7A) and LDH release assays (H2O2 (Figure 7B) and TNBS (Figure 7C)).

[0115] As shown in Figures 7A to 7C, H2O2 and TNBS induced cell death, as indicated by an increase in PI-positive cells and elevated LDH release, respectively. However, in the presence of reduced β-lactoglobulin or GSH, cell death was almost completely prevented. The protective effect of reduced β-lactoglobulin was almost equivalent to that of GSH. The effect of reduced β-lactoglobulin is mediated through the thiol group, and when the thiol group is blocked with maleimide (blocked r-β-LG), the protective effect is largely lost.

[0116] Next, we examined H2O2 or TNBS-induced cellular protein oxidation by reduced β-lactoglobulin.

[0117] Specifically, Caco-2 cells were exposed to 500 μM H2O2 (Figure 7D) or 750 μg / mL TNBS (Figure 7F) for 3 hours in the presence of 5 mg / mL conventional β-lactoglobulin or reduced β-lactoglobulin, and the thiol groups in the resulting cell lysates were quantified by fluorescent maleimide labeling.

[0118] As shown in Figures 7D to 7G, consistent with its cell death prevention effect, reduced β-lactoglobulin, unlike conventional β-lactoglobulin, prevented the reduction of H2O2 and TNBS-induced thiol groups. This indicates a thiol-dependent antioxidant effect. Figure 7E shows the quantitative results of the blot from Figure 7D. Figure 7G shows the quantitative results of the blot from Figure 7F.

[0119] From these results, it was found that reduced β-lactoglobulin prevents H2O2 and TNBS-induced oxidative cytotoxicity, as well as oxidative stress, in cultured colon epithelial cells (Caco-2 cells).

[0120] (Test Example 7: Effects on TNBS and RSL-3-induced ferroptosis in colonic epithelial cells) To further support the important involvement of ferroptosis in TNBS-induced colorectal injury, Caco-2 cells were exposed to 100 μM DFO, 1 μM reproxistatin-1, or 1 mM GSH for 24 hours in or without 1 mg / mL TNBS, and cell death was evaluated by Calcein-AM / PI staining (Figure 8A) and LDH release assay (Figure 8B).

[0121] As shown in Figures 8A and 8B, TNBS-induced cell death was significantly prevented by the ferroptosis inhibitors DFO and reproxistaltin, and the antioxidant GSH, confirming the important involvement of ferroptosis.

[0122] Next, to confirm that reduced β-lactoglobulin has anti-ferroptotic activity, we observed the effect of reduced β-lactoglobulin on RSL-3-induced cell death, a ferroptosis inducer.

[0123] Specifically, Caco-2 cells were exposed to 5 mg / mL conventional β-lactoglobulin or 5 mg / mL reduced β-lactoglobulin for 20 hours in or without 25 μM RSL-3, and cell death was evaluated by Calcein-AM / PI staining (Figure 8C) and LDH release assay (Figure 8D).

[0124] As shown in Figures 8C and 8D, RSL-3-induced ferroptosis cell death was significantly prevented by reduced β-lactoglobulin. These results support the inhibitory effect of reduced β-lactoglobulin on TNBS-induced ferroptosis in cultured cells.

[0125] These results indicate that reduced β-lactoglobulin prevents TNBS and RSL-3-induced ferroptosis in cultured colonic epithelial cells.

[0126] Next, to further support the ferroptosis-preventive effect of reduced β-lactoglobulin, we observed TNBS or RSL-3-induced changes in intracellular iron ion and lipid peroxide formation.

[0127] Caco-2 cells were exposed for 1 hour to 5 mg / mL conventional β-lactoglobulin or 5 mg / mL reduced β-lactoglobulin in the presence or absence of 1000 μg / mL TNBS or 25 μM RSL-3.

[0128] As shown in Figures 9A to 9C, it was found that the increase in intracellular iron ions, induced by TNBS or RSL-3, was completely suppressed by reduced β-lactoglobulin. The effects of these reduced β-lactoglobulins were thiol group-dependent and disappeared when the thiol group was blocked with maleimide. Furthermore, it was found that conventional β-lactoglobulin also exhibits a certain degree of inhibitory effect. Figure 9B is a graph showing the quantitative results of the image in the upper part of Figure 9A. Figure 9C is a graph showing the quantitative results of the image in the lower part of Figure 9A.

[0129] As shown in Figures 9D to 9G, consistent with changes in intracellular iron ions, it was found that the increase in lipid peroxide formation, induced by TNBS (Figures 9D and 9E) or RSL-3 (Figures 9F and 9G), was strongly suppressed by reduced β-lactoglobulin. The effects of these reduced β-lactoglobulins were thiol group-dependent and disappeared when the thiol group was blocked with maleimide. Furthermore, it was found that conventional β-lactoglobulin also exhibits a strong inhibitory effect. Figure 9E is a graph showing the quantitative results of the image in Figure 9D. Figure 9G is a graph showing the quantitative results of the image in Figure 9F.

[0130] From these results, it was found that TNBS induces ferroptotic colonic cell damage in colonic epithelial cells, and reduced β-lactoglobulin strongly prevents this through a thiol group-dependent mechanism.

[0131] (Test Example 8: Effects of bone marrow-derived macrophage cells (cultured macrophages) on TNBS and LPS-induced oxidative stress and cytokine production) The effect of reduced β-lactoglobulin on bone marrow-derived macrophage cells was confirmed. Specifically, we confirmed the preventive effect of reduced β-lactoglobulin on TNBS and LPS-induced oxidative stress and cytokine production in bone marrow-derived macrophage cells.

[0132] Bone marrow-derived macrophage cells were cultured and exposed to 5 mg / mL conventional β-lactoglobulin or 5 mg / mL reduced β-lactoglobulin for 6 hours in or without 250 μg / mL TNBS and 10 μg / mL LPS. The protein SOH levels and P38 activation of the treated cell lysates were analyzed by Western blotting to confirm changes in the cellular oxidation state (Figure 10A).

[0133] As shown in Figures 10A to 10C, stimulation with TNBS and LPS induced oxidative stress in myeloid-derived macrophage cells, as indicated by increased protein sulfenate formation and activation of redox-sensitive P38. These oxidative changes were completely prevented in the presence of reduced β-lactoglobulin. Figure 10B shows the quantitative results of the blot (SOH level) of 10A. Figure 10C shows the quantitative results of the blot (P38 activation) of 10A.

[0134] Next, bone marrow-derived macrophage cells were cultured and exposed to 5 mg / mL conventional β-lactoglobulin or 5 mg / mL reduced β-lactoglobulin for 6 hours in or without 250 μg / mL TNBS and 10 μg / mL LPS. The production of IL-1β and TNFα in the treated bone marrow-derived macrophage cells was measured by ELISA.

[0135] As shown in Figures 10D and 10E, consistent with the prevention of intracellular oxidative stress, reduced β-lactoglobulin strongly inhibited TNBS / LPS-induced IL-1β (Figure 10D) and TNFα (Figure 10E) production in cultured macrophages.

[0136] These results indicate that reduced β-lactoglobulin prevents TNBS and LPS-induced oxidative stress and cytokine production in cultured macrophages.

[0137] In summary, we succeeded in manifesting the antioxidant capacity of β-lactoglobulin derived from whey protein by reductively modifying it. Reduced β-lactoglobulin exhibits potent antioxidant activity by scavenging hydrogen peroxide and reactive oxygen species, and has also been shown to interact with the body's antioxidant system through the -SH / -SS exchange reaction with GSH / GSSG (sometimes referred to as the "SH / SS exchange reaction" or "thiol disulfide exchange reaction"). In animal studies, reduced β-lactoglobulin was found to suppress colonic injury and inflammation and prevent the progression of associated renal impairment in a TNBS-induced colitis model. The mechanism of action was suggested to involve the improvement of oxidative stress and the inhibition of ferroptosis. Furthermore, cell culture experiments confirmed that reduced β-lactoglobulin protects against oxidative stress-induced ferroptosis-induced cell damage. These results demonstrate that revealing the antioxidant capacity hidden within the structural protein β-lactoglobulin through reductive methods and developing it as a novel therapeutic agent for oxidative stress-related diseases is a useful approach. The potential for practical application lies in using inexpensive whey protein-derived raw materials in antioxidant development. This study, which revealed novel aspects of β-LG, is considered a significant achievement in terms of both the effective utilization of dairy-derived resources and the treatment of oxidative stress diseases.

[0138] The results above indicate that β-lactoglobulin derivatives having two or more thiol groups possess significant ferroptosis-inhibiting and antioxidant effects, and can be used as therapeutic compositions for intestinal or kidney diseases.

Claims

1. A ferroptosis inhibitor characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

2. The ferroptosis inhibitor according to claim 1, wherein the β-lactoglobulin derivative having two or more thiol groups does not have a disulfide bond.

3. An antioxidant characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

4. The antioxidant according to claim 3, wherein the β-lactoglobulin derivative having two or more thiol groups does not have a disulfide bond.

5. A therapeutic composition for intestinal or kidney disease, characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

6. A food product characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

7. A cosmetic product characterized by containing a β-lactoglobulin derivative having two or more thiol groups.

8. A method for producing a ferroptosis inhibitor, characterized by including treatment of β-lactoglobulin with a reducing agent.

9. A method for producing an antioxidant, characterized by including treatment of β-lactoglobulin with a reducing agent.

Images